The clinical potential of macromolecule-based neurotherapeutics is currently not realized due to the limited access such drugs gain to the CNS. The long term goal is to develop efficient and specific vascular delivery approaches. Receptor-mediated transport mechanisms at the blood-brain barrier have shown promise for this purpose, but the receptor systems targeted to date (e.g. transferrin or insulin receptor) are not truly brain specific. The objective of this particular application is to establish specific peptide- major histocompatibility (MHC) class I complexes on brain vascular endothelium as viable portals for drug delivery. Peptide-MHC complexes are formed from a vast variety of endogenous peptide fragments and different alleles of MHC class I molecules, and are physiologically recognized by T-cell receptors (TCR). Recently it became possible to generate TCR mimic monoclonal antibodies of high specificity and affinity for distinct human peptide-MHC combinations. TCR mimics hold the potential of tissue / cell type specific targeting. While they have to date primarily been explored as tools for targeting diseased cells, the TCR mimic strategy lends itself principally to target specific complexes expressed by any cell type. The central hypothesis of this work is that TCR mimic antibodies enter brain-derived endothelial cells and thus can be used as delivery vectors. The rationale of the project is to lay the base for the development of highly blood-brain barrier specific diagnostic and therapeutic agents using TCR mimics as vectors. Preliminary studies with one of the TCR mimics indicate that endothelial cells derived from human brain present its corresponding target peptide-MHC combination, and can bind and internalize the antibody. The proposed work will follow two specific aims: 1) Investigate the interaction of TCR mimic antibodies with brain-derived endothelial cells. This will include quantitative binding and uptake studies in vitro and characterization of the subcellular compartments involved, as well as experiments addressing the extent of transendothelial transport (transcytosis). Transgenic mouse strains expressing human MHC class I alleles will be examined to identify suitable in vivo models for follow up projects. 2) Determine the pharmacological activity of a nanoparticulate drug targeted by a TCR mimic. The TCR mimic antibody will be coupled to a DNA/polymer complex, which has previously demonstrated potent anti- inflammatory drug effects when targeted to brain-derived endothelial cells by a transferrin receptor antibody. Endothelial cell cultures will receive inflammatory stimuli and the effect of drug targeted by the TCR-mimic will be measured by quantitative RT-PCR of inflammatory markers and by a functional assay (monocyte adhesion). The approach is innovative, because TCR mimics have not been previously considered as vectors for targeting brain. The proposed research is significant, because it can pave the way to a novel vascular targeting strategy, with potential applications reaching from CNS disorders to diseases of other vascularized organs, and tumors. PROJECT NARRATIVE: The proposed studies address an important area of research concerned with the development of better drugs to treat diseases of the brain. The findings can have wide implications for potential protein- or DNA-based therapeutics, which are often hampered by their inability to reach the target tissues.